Fungal Samples Research Articles

Issues with percent coverage-based non-viable fungal analysis and new methodology for enumeration-based analysis

The choices of methodology for analysis of non-viable fungal surface samples are yet to be agreed upon. The two main types of analysis of non-viable fungal (gross fungi) samples can be generally categorised as counting based (enumeration of fungal structures) or categorization based (such as percent coverage estimates or fungal rating categories). We present evidence of flaws in the percent coverage methodology due to a strong dependence on individual analyst’s subjective estimates. By using image analysis and a survey of analysts we show the high variability between analysts’ percent coverage estimates. We also propose an enumeration-based methodology which attempts to address problems in current counting-based methods by using a semi-random grid pattern of fields of view rather than a traverses-based approach.

Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters.

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.

Visible label-free detection of bacterial DNA using flocculation of sterically stabilised cationic latexes.

The current gold standard diagnostic for bacterial infections is the use of culture, which can be time consuming and can take up to five days for results to be reported. There is therefore an unmet clinical need for a rapid and label free alternative. This paper demonstrates a method of detecting the presence of amplified DNA from bacterial samples using a sterically-stabilised, cationic polymer latex and widely available equipment, providing an accessible alternative DNA detection technique. If DNA is present in a sample, successful amplification by polymerase chain-reaction (PCR) results in the amplified DNA inducing flocculation of the polymer latex followed by rapid sedimentation. This results in a visible and obvious change from a milky-white dispersion to a precipitated latex with a colourless and transparent supernatant, thus giving a clear visual indication of the presence or absence of amplified DNA. Specifically, the response of four polymer latexes with different morphologies to the addition of amplified bacterial DNA was investigated. Cationic latexes flocculated rapidly whereas non-ionic and anionic latexes did not, as judged by eye, disc centrifuge photosedimentometry (DCP), and UV-visible spectrophotometry. The stability of several cationic latexes with different morphologies in typical PCR reagents was investigated. It was found that unwanted flocculation occurred for a latex with a non-ionic core and a cationic corona (poly[2-vinyl pyridine-b-benzyl methacrylate], prepared by polymerisation-induced self-assembly) whereas a ∼700 nm PEGMA-stabilised P2VP latex (non-ionic stabiliser, cationic core), prepared by emulsion polymerisation remained stable. The sensitivity and rate of sedimentation of the PEGMA-stabilised P2VP latex was demonstrated by varying the sequence length and concentration of amplified DNA from Pseudomonas aeruginosa using universal bacterial primers. DNA concentrations as low as 0.78 ng μl-1 could readily be detected within 30 minutes from the addition of amplified DNA to the latex. Furthermore, the specificity of this method was demonstrated by showing a negative result occurs (no flocculation of the latex) when PCR product from a fungal (Candida albicans) sample using bacterial primers was added to the latex.

Airborne pollen and fungi indoors: Evidence from primary schools in Lithuania

The number of children suffering from respiratory allergies and asthma has been increasing worldwide and, hence, it is crucial to understand the burden of inhalant biological particles present in school facilities, where children spend one third of their life. From the perspective of indoor air quality, while there are numerous studies on outdoor bioaerosol exposure, there are still uncertainties regarding the diversity and deposition of airborne pollen and fungi indoors. When it comes to schools, there is limited research as to the potential bioaerosol exposure. Here we studied the indoor environment of public schools aiming to reveal whether primary schools of different sizes and at localities of different levels of urbanization may exhibit a variability in the biodiversity and abundance of particles of biological origin, which could pose a risk to child health. To achieve this, 11 schools were selected, located in a variety of environments, from downtown, to city centre-periphery, and to the suburbs. Fungal and pollen samples were collected from various surfaces in school classrooms and corridors, using passive air sampling and swab sampling. We demonstrated that fungi and pollen are detected in school premises during and after the vegetation season. The highest diversity of bioaerosols was found on the top of cabinets and windowsills, with Penicillium, Cladosporium and Acremonium being the most abundant indoors. The levels of fungi were higher in schools with more students. The diversity and amount of pollen in the spring were significantly higher than in samples collected in autumn. Our findings complemented existing evidence that bioaerosol measurements in schools (including kindergartens or informal education facilities) are vital. Hence, we here suggest that, in addition to monitoring air quality and bacterial levels indoors, fungi and pollen measurements have to be integrated in the existing regular biomonitoring campaigns so as to prevent exposure, increase awareness and manage efficiently allergic symptomatology.

Seasonal and spatial variations of bioaerosols and antibiotic resistance bacteria in different wards of the hospital

Introduction: Transmission of bioaerosols through the air is known as an important route for a wide range of nosocomial infections. Therefore, in the present study, we aimed to evaluate the type and diversity of bioaerosols and antibiotic resistance of bacterial bioaerosols in the indoor environments of Sina educational and treatment hospital, Tabriz, Iran. Methods and materials: 150 samples of bacteria and fungi (75 fungi and 75 bacteria) bioaerosol samples were collected on petri dish containing Sabouraud dextrose agar from February to March and June to July 2020 in three periods of daytime (morning, noon and evening) according to National Institute for Occupational Safety and Health (NIOSH 0-800) standard. After sampling, fungal and bacterial samples were incubated and the disk diffusion agar method (Kirby-Bauer) was used for assessing the antibiotic resistance. Results: The concentration of bioaerosols varied significantly in different wards. In addition, the concentration of bioaerosols in winter was observed to be higher than in summer. The highest and lowest airborne fungal concentrations were found in burns operating room and men's infectious ward (49 CFU/m3) and children's burns ward (28 CFU/m3), respectively. The predominantly isolated bacteria were Streptococcus spp. (38%) and Staphylococcus spp. (37%). Also, the main isolated fungi belonged to the genera Aspergillus (75.9%) and Penicillium (22.5%). The highest rates of antibiotic resistance were observed for colistin (100%) in Gram-negative and penicillin (84.2%) in Gram-positive. Conclusion: Timely and regular disinfection of hospital wards can affect the density of bioaerosols. Owing to the prevalence of COVID-19 epidemic in the world, the staff and patients often were wearing masks, gloves and special clothing as well as using disinfectants to prevent coronavirus infection in wards during the summer sampling.

Microbiome Analysis of the Bamboo Aphid Melanaphis bambusae Infected with the Aphid Obligate Pathogen Conidiobolus obscurus (Entomophthoromycotina).

Insect-associated microbes exert diverse effects on host fitness. This study provides insights into the microbiota of the bamboo aphid, Melanaphis bambusae, and their response to Conidiobolus obscurus infection. 16S rRNA and ITS sequencing data were used to analyze the bacterial and fungal samples associated with healthy, infected, and starved aphids. At ≥97% nucleotide similarity, the total reads were clustered into 79 bacteria and 97 fungi operational Taxonomic Units (OTUs). The phyla Proteobacteria and Ascomycota dominated the bacterial and fungal communities, respectively. The significant divergence in OTU distribution presented differential profiles of the microbiota in response to host conditions. Lower α-diversity indices were found in bacterial and fungal diversity when the aphids were experiencing fungal infection and starvation stresses, respectively. The β-diversity analyses of the communities showed significant differences among the three host conditions, demonstrating that aphid-associated microbiota could significantly shift in response to varying host conditions. Moreover, some OTUs increased under fungal infection, which potentially increased aphid susceptibility. Presumably, C. obscurus infection contributed to this increase by causing the disintegration of host tissues other than host starvation. In conclusion, understanding the differentiation of aphid microbiota caused by fungal entomopathogens helped facilitate the development of novel pest management strategies.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ FA) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers. First, the native polymers were isotopically N-acetylated, then enzymatically hydrolyzed to A and R (2H3N-acetyl-d-glucosamine – former D) monomers. Relative abundances of A and R units were used to calculate x̅ FA, and a double-isotopically labeled internal standard R* ([13C2,2H3] N-acetyl-d-glucosamine) monomer was used to calculate the absolute amounts of GlcNAc and GlcN units present in the fungal samples. The method was validated using known chitosan polymers and is suitable for both purified cell walls and whole mycelia.

Bioinspired Hydrophilic and Oleophilic Absorption Media from Biotemplated Fungi

Fungi are an incredibly diverse biological kingdom with organisms that have a wide range of morphologies, properties, and structures. Previous research has investigated the use of filamentous fungi, which have naturally porous structures created by hyphal filaments, as a means of phytoremediation. This study uses these natural fungal structures to create bioinspired materials that capture both the structure and functional absorption properties of fungi. Three types of filamentous fungi with different hyphal structures (monomitic, dimitic, and trimitic) are used as organic templates to create inorganic media using two different biotemplating methods to create silica and hybrid samples. Characterization of these samples is completed using scanning electron microscope imaging, chemical characterization, nanoindentation, and hydrophilic and oleophilic absorption tests. Biotemplated samples have similar structures as their organic templates, but contained silica, which is not present in natural, dehydrated fungal samples. Fourier transform infrared analysis shows better cross‐linking in the hybrid samples, which also have higher mechanical resistance than the silica samples. Absorption testing demonstrates that silica samples are closest to mimicking the absorption properties of natural, dehydrated samples. Of the three hyphal structures, the monomitic samples show the greatest increase in mechanical properties and maintenance of absorption properties when biotemplated.

Excitation-Dependent Fluorescence Helps to Indicate Fungal Contamination of Aquatic Environments and to Differentiate Filamentous Fungi

Fungal contamination of aquatic environments can lead to an adverse impact on the environment and human health. (1) The search for fast, inexpensive and appropriate methods for detection of fungi is very moving rapidly due to their significant impact on ecosystem functions and human health. (2) We focused on examination of fluorescence proxies able to distinguish chromophoric matter occurring in different fungi. Spectroscopic studies were performed on five strains of filamentous fungi: Trichoderma harzianum, Fusarium solani, Alternaria alternata, Cladosporium cladosporioides and Aspergillus terreus. (3) The results showed that most of the fungal autofluorescence was emitted by amino acids, melanin-like compounds, NAD(P)H and flavins. The spectra of five fungal species cultivated as planktonic or surface-associated forms turned out to be different. Protein fluorescence can be used to detect general microbial contamination. Presence of excitation wavelength dependent mode and the “blue shift” of fluorescence (emission bands 400–500 nm) can be suggested as specific feature of fluorescence of fungal melanin-containing samples. (4) The determination based on fluorescence spectra obtained at a certain excitation/emission wavelengths pair and at whole excitation-emission matrices (EEMs) coupled to principal component analysis (PCA) algorithms as a tool of improving detection capabilities can be suggested to enable fast and inexpensive monitoring of fungal contamination of aquatic environments.

Surface-enhanced Raman spectral characterization of antifungal activity of selenium and zinc based organometallic compounds

Surface-enhanced Raman spectroscopy (SERS) is used to identify the biochemical changes associated with the antifungal activities of selenium and zinc organometallic complexes against Aspergillus niger fungus. These biochemical changes identified in the form of SERS peaks can help to understand the mechanism of action of these antifungal agents which is important for development of new antifungal drugs. The SERS spectral changes indicate the denaturation and conformational changes of proteins and fungal cell wall decomposition in complex exposed fungal samples. The SERS spectra of these organometallic complexes exposed fungi are analyzed by using statistical tools like principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA). PCA is employed to differentiate the SERS spectra of fungal samples exposed to ligands and complexes. The PLS-DA discriminated different groups of spectra with 99.8% sensitivity, 100% specificity, 98% accuracy and 86 % area under receiver operating characteristic (AUROC) curve.

P420 Fungal detection by means of HCR using 2D-Covalent Organic Framework Nanosheet

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe timely diagnosis of fungal infections is of prime importance for prescribing appropriate anti-fungal drugs. Current methods for fungal diagnosis involve culture-based methods, antibody-based detection using lateral flow assays and RT-PCR. In the present work, we devised a non-enzymatic amplification using 2D-Covalent Organic Framework (COF) nanosheet for the detection of fungal DNA.Objectives(1) Validation of exfoliated 2D COF Nanosheet as an efficient DNA detection tool via Hybridization chain reaction (HCR) triggered fluorescent assay. (2) Sequence retrieval and probe generation of fungal sample and detection of extracted target DNA via fluorescent assay.MethodA novel COF was synthesized and characterization was done using FTIR, BET, TGA, XRD, and SEM. Probes for the detection of fungi (Candida, Aspergillus, and Mucor) were designed using NUPACK software. HCR was monitored for different time and probe concentrations and standardized reaction was used for the detection of fungal RNA.ResultsFTIR, BET, TGA, XRD, and SEM confirmed the structure and formation of COF-nanosheet. H1, H2 probes at a concentration of 1μm and in presence of Target DNA (0.001μm-1μm) showed HCR reaction at 1.5 h. Fluorescence quenching was observed when probes were mixed with both, bulk COF and COF-nanosheets but increased quenchingConclusionFungal detection can be done by means of HCR using the Covalent nanosheets.

First record of the occurrence of Pleurotus species on new hosts in Bangalore, Karnataka, India

Mushrooms have wide geographical distribution, with greatest commercial importance, both in temperate areas and tropical regions of the world. The agarics mycota of tropical eco-regions of Bangaluru, Karnataka was surveyed in different seasons from 2019 to 2020 for collection and identification of fungal samples. Detailed macroscopic and microscopic study of fungal samples was identified as Pleurotus pulmonarius (Fr.) Quél. P. ostreatus (Jack.) P. Kumm. P. populinus O. Hilber & O. K. Mill. For the first time, P. pulmonarius (Fr.) Quél was causing stem decay in Mangifera indica L. was reported from India. New host record of P. populinus O. Hilber & O. K. Mill on Spathodea campanulata was reported from India. The 3 species of Pleurotus was reported for the first time from the study area of Bengaluru, Karnataka, India.

Airborne fungal and bacterial microbiome in classrooms of elementary schools during the COVID-19 pandemic period: Effects of school disinfection and other environmental factors.

The aim of the study was to examine the effects of environmental factors including disinfection on airborne microbiome during the coronavirus disease 2019 pandemic, we evaluated indoor and outdoor air collected from 19 classrooms regularly disinfected. Extracted bacterial and fungal DNA samples were sequenced using the Illumina MiSeq™ platform. Using bacterial DNA copy number concentrations from qPCR analysis, multiple linear regressions including environmental factors as predictors were performed. Microbial diversity and community composition were evaluated. Classrooms disinfected with spray ≤1 week before sampling had lower bacterial DNA concentration (3116 DNA copies/m3) than those >1 week (5003 copies/m3) (p‐values = 0.06). The bacterial DNA copy number concentration increased with temperature and was higher in classrooms in coastal than inland cities (p‐values <0.01). Bacterial diversity in outdoor air was higher in coastal than inland cities while outdoor fungal diversity was higher in inland than coastal cities. These outdoor microbiomes affected classroom microbial diversity but bacterial community composition at the genus level in occupied classrooms were similar between coastal and inland cities. Our findings emphasize that environmental conditions including disinfection, climate, and school location are important factors in shaping classroom microbiota. Yet, further research is needed to understand the effects of modified microbiome by disinfection on occupants' health.

Meta-analysis of fungal plant pathogen Fusarium oxysporum infection-related gene profiles using transcriptome datasets.

Fusarium oxysporum is a serious soil-borne fungal pathogen that affects the production of many economically important crops worldwide. Recent reports suggest that this fungus is becoming the dominant species in soil and could become the main infectious fungus in the future. However, the infection mechanisms employed by F. oxysporum are poorly understood. In the present study, using a network meta-analysis technique and public transcriptome datasets for different F. oxysporum and plant interactions, we aimed to explore the common molecular infection strategy used by this fungus and to identify vital genes involved in this process. Principle component analysis showed that all the fungal culture samples from different datasets were clustered together, and were clearly separated from the infection samples, suggesting the feasibility of an integrated analysis of heterogeneous datasets. A total of 335 common differentially expressed genes (DEGs) were identified among these samples, of which 262 were upregulated and 73 were downregulated significantly across the datasets. The most enriched functional categories of the common DEGs were carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Nine co-expression modules were identified, and two modules, the turquoise module and the blue module, correlated positively and negatively with all the infection processes, respectively. Co-expression networks were constructed for these two modules and hub genes were identified and validated. Our results comprise a cross fungal-host interaction resource, highlighting the use of a network biology approach to gain molecular insights.

Plain language summary: Does a person's age affect how common fungal infections are and how well drugs can kill the infections?

Fungi are types of microbes that include molds and yeasts. Fungal infections can make people ill and can even cause death, especially in older people. They can be treated using antifungal drugs, but some fungi are drug resistant. This means the drug cannot kill the fungi. This is a summary based on a study that looked at fungal samples to find out more about antifungal drug resistance in adults younger than 65 compared with adults aged 65 and older. The study found that one type of drug-resistant fungus, called Candida parapsilosis, was more common in older people than in younger people. Another type, called Aspergillus fumigatus, was more common in younger people than in older people. We also found genetic changes in drug-resistant fungi. These changes could explain why the drugs did not work. We hope that the findings from this study can help scientists create new treatments for drug-resistant fungal infections.

The mysterious mould outbreak - A comprehensive fungal colonisation in a climate-controlled museum repository challenges the environmental guidelines for heritage collections

• The xerophilic Aspergillus halophilicus has colonised a museum repository. • It was climate-controlled; the growth was not detectable with traditional methods. • Fungal growth in museum collections threatens the preservation of cultural heritage. • The massive fungal infestation may cause occupational exposure. • Guidelines for heritage collections do not consider the risk of xerophilic fungi. Within the last decade, fungal colonisations have increased in Danish museum repositories. The growth is unexpected, as many Danish museums strive to comply with the environmental guidelines for heritage collections. When fungal growth develops in heritage collections, it threatens the heritage preservation and occupational health of museum staff. Therefore, it is crucial to prevent. This study characterised a fungal colonisation in a 1450m 2 museum repository, striving to meet the guidelines for heritage collections with relative humidity below 60%. After fungal discovery, the repository was examined twice by an environmental laboratory with morphological identification of fungi and quantification of fungal biomass based on fungal enzyme activity. However, the reports were not sufficient to qualify a recovery process. A research study with a broader approach was conducted to further elucidate the problem. The study included 1) building examination, 2) fungal surface sampling and morphological ID, 3) ID of fungal isolates with DNA sequencing, and 4) activated fungal air sampling and morphological ID. Although the relative humidity was measured to meet the guidelines for heritage collections with no evidence of moisture or microclimate, hyaline and white fungal colonies were distributed on heritage artefacts throughout the repository. There was no growth on interior and building structures. Cultivation of air samples on DG18-agar and V8®agar showed the presence of common indoor fungi, while artefact samples cultivated on the same media showed no growth. In contrast, cultivation of air samples and artefacts samples on the low water activity agar MY50G followed by DNA-sequencing showed high concentrations of the xerophilic fungi A. halophilicus, A. domesticus, A. magnivesiculatus and A. vitricola , belonging to Aspergillus section Restricti . These fungi are characterised by growing at low water activity corresponding to low relative humidity. The museum repository seemed to provide this environment with relative humidity below 60%. The study emphasised that examining the same fungi using different approaches may obtain very different results. Furthermore, the study questioned if the environmental guidelines for heritage collections adequately prevent the risk of xerophilic fungal growth. Xerophilic fungi are not adequately included in the risk assessment underlying the preventive conservation framework. Consequently, the risk is not included in the revised environmental guidelines accepting RH between 40-60% to support more sustainable heritage storage. It has not been studied if the revision would increase the risk of xerophilic fungal growth before it was accepted and implemented. This study indicates that it could be the case. Close collaboration between mycologists and museum professionals may develop more standardised and targeted detection and prevention practices for heritage repositories. The risks of xerophilic fungal growth should be included in the preventive conservation framework ensuring heritage preservation and occupational health of museum staff.

Identification and Stratification of Cultured Microorganisms from Wind Cave

Wind Cave National Park in the Black Hills of South Dakota contains many fungal, bacterial, and archaeal organisms. These biotic communities tend to be low in diversity and sensitive to changes in external inputs of organic matter from seasonal snow melts and tourist traffic. Tourist traffic in particular can affect the natural state of the cave environment and alter species composition and population densities of the resident biota. The goal of this project was the molecular identification of the various fungal and bacterial samples between remote and public sites of Wind Cave to better understand the impact of tourist traffic on cave biota. DNA obtained from cultured samples was used to perform PCR‐based sequencing analysis using primers targeting either a specific region of the 16S small ribosomal subunit for identifying bacteria or an internal transcribed spacer (ITS) for identifying fungal lineages. Phylogenetic trees were created of both the fungal and bacterial genra stratified by cave location.

Morpho-molecular characterisation of Arecophila, with A.australis and A.clypeata sp. nov. and A.miscanthi comb. nov.

Three arecophila-like fungal samples were collected on dead culms of gramineous plants in China. Morphological studies of our new collections and the herbarium specimen of Arecophilagulubiicola (generic type) were conducted and the morphological affinity of our new collections with Arecophila was confirmed. Maximum likelihood and Bayesian analyses using combined ITS, LSU, rpb2 and β-tubulin data from our collections revealed the phylogeny of Cainiaceae. The monospecific genus Alishanica (type species Al.miscanthi), which had been accepted in Cainiaceae, is revisited and synonymised under Arecophila. Based on morphology and phylogeny, Arecophilaaustralis sp. nov. and A.clypeata sp. nov. are introduced as new species, while A.miscanthi is a new record for China. All the new collections are illustrated and described.

Evaluation of the Vitek Ms system for the identification of filamentous fungi.

The use of MALDI-TOF for fungal identification remains a challenge. In this study, using a commercial protein extraction kit and updated database, VITEK MS system was able to identify up to 89.0% of a diverse collection of 200 filamentous fungi representing 43 fungal species.

Citrus bergamia: Kinetics of Antimicrobial Activity on Clinical Isolates.

Background: The inappropriate use of antibiotics has increased selective pressure and the spread of multi-drug-resistant (MDR) pathogens, which reduces the possibility of effective treatment. A potential alternative therapeutic approach may be represented by essential oils, such as the distilled extract of bergamot (Citrus bergamia Risso et Poiteau). Such natural products exercise numerous biological activities, including antimicrobial effects. Methods: This work aimed to evaluate the kinetics of the bactericidal and fungicidal activity of the distilled extract of bergamot on MDR bacteria and fungi from clinical specimens using the time-kill assay. Furthermore, the antimicrobial activity of the distilled extract of bergamot on the morphology and cellular organization of clinical pathogens was evaluated by confocal laser scanning microscopy. Results: Our results demonstrated that the distilled extract of bergamot exhibited significant antimicrobial activity and a specific bactericidal effect against the bacterial and fungal strains tested. Furthermore, confocal microscope images clearly showed compromised membrane integrity, damage and cell death in bacterial samples treated with the distilled extract of bergamot. In addition, progressive alterations in cell-wall composition, cytoplasmic material and nucleus structure triggered by exposure to the distilled extract of bergamot were identified in the fungal samples considered. Conclusions: Our data suggest that the use of essential oils, such as distilled extract of bergamot (Citrus bergamia Risso et Poiteau), can represent a valid alternative therapeutic strategy to counteract antibiotic resistance of pathogens.